Monoclonal antibodies to feline-t-lymphotropic lentivirus

ABSTRACT

Monoclonal antibodies specific for an epitope of an FIV-encoded antigen.

This invention was made with government support under Grant AI-27605awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

This application is a continuation-in-part of O'Connor et al., U.S. Ser.No. 279,989, filed Dec. 5, 1988, now abandoned, entitled "MonoclonalAntibodies To Feline-T-Lymphotropic Lentivirus."

This invention relates to monoclonal antibodies to feline-T-lymphotropiclentivirus (also called feline immunodeficiency virus, FIV) and to useof these monoclonals for detection and purification of the virus.

Pedersen et al., 235 Science 790, 1987, described detection andisolation of FIV from domestic cats having an immunodeficiency-likesyndrome. The virus was purified by centrifugation on sucrose gradientsin Tris base pH 7.4 containing 0.1 M NaCl and 1 mM EDTA. Western blotsprepared from gradient purified virus and their reaction with sera fromexperimentally infected cats was determined. A few protein bands weredetected and "though antigenic comparison was not made, the position ofthese bands may correspond to the major core protein p24, qaq precursorprotein p55 and endonuclease protein p32 of HIV".

SUMMARY OF THE INVENTION

In a first aspect, the invention features monoclonal antibodies specificfor an epitope of an FIV-encoded antigen, e.g., a protein, polypeptide,or glycoprotein. By monoclonal antibody is meant any antibody that isproduced from a single antibody producing cell which has been cloned toproduce an antibody producing cell line. It does not include the variousantibodies found in a polyclonal preparation, i.e., a preparationproduced by inoculation of an animal with an antigen and recovery of theresulting serum. By epitope is meant any specific amino acid sequence,modified amino acid sequence, or protein secondary or tertiary structurewhich is recognized by an antibody. This epitope may be present on oneor more antigens of FIV. but is not present in any of the othercomponents commonly associated with FIV, for example, feline cells andserum.

In preferred embodiments, the epitope is present on an antigen selectedfrom the group consisting of the p10, p15, p26, p47, p110, gp40 or gp130antigens of FIV (i.e., those FIV-encoded antigens with molecular weightsof 10, 15, 26, 40, 50, 110 or 130 kD as described below); the antibodyhas sufficient affinity for the epitope to selectively identify theepitope in an immunoassay for FIV; the antibody recognizes an epitope onthe FIV viral particle; the antibody is conjugated with a detectablelabel, the label not significantly interfering with the specificity oraffinity of the antibody for the epitope, most preferably the label isan enzyme, e.g., horseradish peroxidase; and the antibody is produced bya cell line deposited with the American Type Culture Collection, andassigned the number HB9888, HB9889, or HB9890.

In a second aspect, the invention features a solution containing aplurality of the above monoclonal antibodies, each antibody beingspecific for an epitope of an FIV-encoded antigen.

In a third aspect, the invention features a method for detection of anepitope of an FIV-encoded antigen in a sample, including the steps ofproviding a monoclonal antibody specific for the epitope; contacting theantibody with the sample under conditions in which the antibody forms acomplex with the epitope; and detecting the complex, wherein thepresence of the complex indicates the presence of the epitope in thesample.

This invention provides monoclonal antibodies suitable for use in assaysand purification of FIV. Applicant has determined a method by whichsufficiently pure FIV antigen can be prepared in order to allow thesemonoclonal antibodies to be isolated, and to detect the presence of suchmonoclonal antibodies in a screening procedure. Applicant has alsodescribed means by which useful antibodies can be distinguished fromthose which are not useful in the invention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The drawings will first briefly be described.

DRAWINGS

FIG. 1 is a photograph of the major viral associated proteins of FIVidentified by polyacrylamide gel electrophoresis (PAGE) and stained withCoomassie Blue R250 (lane A); molecular weight standards are shown inlane B; and

FIG. 2 is a photograph of a Western immunoblot analysis of antibodies toFIV found in serum from cats identified as positive by an ELISA assayfor FIV antibodies;

FIV Antigen

The FIV antigen is derived from FIV-infected cells, and takes the formof FIV virus particles, or is derived from FIV virus particles, asfollows. (Synthetic antigens are also suitable in the assay, as aresubstantially purified polypeptides derived from FIV particles.) Masterseed virus producing cultures were obtained in the form of a continuousfeline cell line infected with FIV isolate #2427 (CRFK-FIV or Petalumastrain) from Dr. Niels Pedersen (University of California, Davis,Calif.). The parent cell line is Crandell feline kidney cellpersistently infected with FIV. The cell line was deposited with theAmerican Type Culture Collection on Jul. 13, 1988 and assigned thenumber CRL9761. Applicants and their assignees acknowledge theirresponsibility to replace this culture should it die before the end ofthe term of a patent issued hereon, 5 years after the last request for aculture, or 30 years, whichever is the longer, and its responsibility tonotify the depository of the issuance of such a patent, at which timethe deposit will be made available to the public. Until that time thedeposit will be made available to the Commissioner of Patents under theterms of 37 C.F.R. §1-14 and 35 U.S.C. §112.

Other virus cultures can be obtained as described by Pedersen, supra, orby Harbour et al., 122 The Veterinary Record 84, 1988. Seed stocks ofvirus producing cell cultures were obtained by freeze downs of FIVinfected master seed cell cultures following at least 19 post infectionpassages in culture. Additional seed stocks of virus producing cultureswere obtained by either infection of the continuous feline cell culturewith FIV master seed virus or by single cell microwell cloning of highlevel FIV producers from the original FIV infected master seed cellculture. For propagation, master seed virus infected feline cellcultures were inoculated into tissue cell culture flasks. Followinggrowth to a confluent monolayer of cells, tissue culture fluid washarvested at intervals of 2-5 days.

Working seed virus was produced by propagation by the master seed cellline permanently infected with FIV. An inoculum was added to tissueculture flasks, in Dulbecco's Modified Eagles medium containing 2 mML-glutamine and 4.5 g per liter/glucose (DME) containing 100 units perml. penicillin and streptomycin and 2 mM glutamine. An inoculum wasadded to tissue culture flasks, incubated, and the spent tissue culturefluid harvested when the cells were grown to confluence. The cells werereleased from the culture vessel with trypsin/EDTA and diluted between1:5 and 1:25 (typically 1:8) in medium. Typically the flasks wereincubated at 36° C.-38° C. for a maximum of 7 days (between 3 and 7days) before fluid and cell harvest. The harvested fluid, including cellmaterial, was centrifuged in a high speed centrifuge (Sorval RC-5B orBeckman J2-2l) leading to separation of supernatant and cell pelletmaterial. The cell pellet was discarded, and the supernatant culturefluid used to prepare working virus. The clarified supernatant was made0.5 M in NaCl and 4%-10% (usually 7%) in polyethylene glycol (PEG 8000,Sigma). Following overnight incubation at 2° C.-7° C., virus waspelleted (at 13,000× g for 30 min.) and resuspended in buffer (10 mMTris, pH 7.6 300 mM NaCl, 1 mM EDTA, at 2° C.-7° C.). After overnightincubation the virus was centrifuged at 13,000 × g for 15 min., thepellet discarded and the supernatant centrifuged on a 50%/80%discontinuous glycerol step gradient in 10 mM Tris 300 mM NaCl, 1 mMEDTA at pH 7.6. Centrifugation was at 100,000× g for 3 hrs. at 4° C. andthe FIV viral band at the 50% -80% interface collected. The band wassuspended in 10 mM Tris, 0.3 M NaCl and 1 mM EDTA and diluted 1:3 in thebuffer and repelleted at 100,000× g for 1 hr. The resulting pellet waspurified virus and was resuspended in the above buffer and stored at-70° C. The resulting virus was substantially free from FIV host cellproteins and was composed of at least 5% p26 (the major nucleocapsidprotein, as measured by densitometric scans of Coomassie Blue 250stained SDS/PAGE as total protein). Such antigen is sufficiently purefor preparation of monoclonal antibodies and for screening for suitablemonoclonal antibodies.

Such purified antigen may be obtained by other techniques, however,applicants have found that high molecular weight contaminants present invirus preparations may be eliminated by use of the high salt (i.e.,greater than physiological range salt concentration) used in thegradient centrifugation procedure.

FIV Glycoprotein

In order to prepare monoclonal antibodies to viral glycoproteins,antigenic glycoprotein was firstly prepared as follows.

Actively growing CRFK FTLV infected cells were scraped from rollerbottles, gently washed with phosphate-buffered saline (PBS), andpelleted. The cell pellet was gently resuspended in 10 mM sodiumphosphate, pH 7.2, at a ratio of 1 ml buffer to 0.1 ml of cell pellet.This suspension was incubated on ice or refrigerated for 5-10 min.,vigorously vortex mixed for 30 seconds, and four volumes of PBS with 1mM PMSF added. The mixture was then vigorously homogenized for 90-120seconds with a Brinkmann Homogenizer PT10/35 with a PTA 20 generator.

The resulting homogenate was clarified for 20 minutes at 5,000 g. Thesupernatant fraction was discarded and the cell membrane pelletresuspended in PBS +0.2% Triton X-100 at a ratio of 2.5 ml buffer to 0.1ml original cell pellet. The mixture was then vigorously homogenized for90-120 seconds with a Brinkmann Homogenizer PT 10/35 with a PTA 20generator. The resulting homogenate was clarified at 100,000 g for 1 hr,the supernatant decanted off and batch bound overnight at 20°-23° C. onPharmacia Lentil Lectin Sepharose 4B at a ratio of 6 ml of resin to 5 mlof original cell pellet.

The Lentil Lectin Suspension was poured through a column, the resincollected, and washed with 15 column volumes of PBS +0.2% Triton X-100.The glycoproteins were then eluted from the resin by subjecting theresin to 5-10 column volumes of PBS +0.2% Triton X-100+200 mM methyl α-Dmannopyranoside, collecting fractions of 1 column volume/tube.

The isolation of glycoproteins was verified by 9% SDS-PAGEelectrophoresis, and checked using ³⁵ S-radiolabeled cell preparationsin conjunction with RIPA data.

Further purification of viral glycoprotein from host cell glycoproteinincludes use of the HPLC or use of a polyclonal antibody for affinitychromotography.

Preparation of FIV Monoclonal Antibodies

Balb/CJ (Jackson Labs) mice were immunized with an initial injection of50 micrograms of FIV antigen per mouse mixed 1:1 with Difco Bactoadjuvant complete. After two weeks a booster injection of 100 microgramsof FIV antigen was injected into each mouse intravenously withoutadjuvant. Three days after the booster injection a fusion was performedwith mouse myeloma cell lines FO or p3X63-Ag8.653. Mid log phase myelomalines were harvested on the day of fusion and checked for viability. Thecells were spun at 300× g for 8 min., separated from the growth medium,and resuspended in serum free DME.

For fusion, an FIV inoculated mouse was killed by cervical dislocationand the spleen aseptically removed. The spleen was washed three times inserum free DME and placed in a sterile Petri dish containing 20 mls ofcomplete medium (DME containing 20% bovine fetal serum, 100 units perml. of penicillin and streptomycin, and 1 mM sodium pyruvate). Torelease cells, the spleen was perfused with a 23 gauge needle.

Cells were placed in a 50 ml conical centrifuge tube and pelleted at300× g for 8 min. The pellet resuspended in 5 ml of 0.17 M ammoniumchloride and placed on ice for 8 min. 5 ml of bovine fetal serum (20%)was added and the cells pelleted again at 300× g for 8 min. Afterresuspension in 10 ml DME the cells were counted and the spleen andmyeloma cells mixed in a ratio of 3:1. The cell mixture was pelleted at200× g for 10 minutes, the supernatant decanted, and the pellet allowedto stand for 5 min. Over a period of 1 min., 1 ml of 50% PEG (PEG 1500mixed 1:1 with Hepes pH 8.1) at 37° C. was added. After 1 min.incubation at 37° C., 1 ml of DME was added over a period of another 1min. and then a second 1 ml of serum free medium added over a period of1 min. Finally, 10 mls of DME was added over a period of 2 min., thecells pelleted at 200× g for 8 min., and the pellet resuspended incomplete medium containing 0.016 mM thymidine, 0.1 mM hypoxanthine, 0.5micromolar aminopterin, and 10% hybridoma cloning factor (1× HAT). Thecells were plated into 96-well plates.

After 3, 5 and 7 days half of the medium in the fusion plates wasremoved and replaced with fresh 1× HAT. After 11 days the hybridoma cellsupernatant wa screened by an ELISA test. In this test, 96 well plateswere coated with FIV antigen by standard technique. One hundredmicroliters of supernatant from each well was added to a correspondingwell on a screening plate and incubated for 1 hr. at 20°-22° C. Afterincubation, each well was washed three times with distilled water and100 microliters of a horseradish peroxidide conjugate of goat anti-mouseIgG (H+L), A, M (1:1500 dilution) was added to each well and incubatedfor 1 hr. at 20°-22° C. After three washes with distilled water, thesubstrate OPD/hydrogen peroxidide was added and incubation continued forfive to fifteen minutes. One hundred microliters of a stop solution (1 Mhydrochloric acid) was then added and the absorbance at 490 nm read.Cultures which had an optical density reading greater than the controlwells were removed to 2 cm² culture dishes, with the addition of normalmouse spleen cells in 1× HT medium. After a further three days all ofthe 2 cm² cultures were rescreened for antibody and those testingpositive again were cloned by limiting dilution. The cells in each 2 cm²culture were counted and cell concentration adjusted to 1×10⁵ cells perml. The cells were diluted in complete medium and normal mouse spleencells at concentrations of hybridoma cells of 5, 10 and 50 cells per mladded. The cells were plated into 96-well plates for each dilution.After 10 days the cloning plates were screened for growth. About 37% ofall wells showed growth. The growth-positive wells were screened forantibody and those testing positive expanded to 2 cm² cultures andprovided with normal mouse spleen cells. The cloning procedure wasrepeated 2 times until a stable antibody-producing hybridoma wasobtained. At this point the cell culture was expanded from 2 to 9 to 75to 150 cm² culture vessels, at which point ascite production could becommenced.

For ascites production, pristane primed IRCFl female mice were used. 0.5ml of pristane was injected intraperitoneally (IP) to each mouse, andthe mouse allowed to rest for 10-60 days. At this time 4.5×10⁶ cellswere injected IP into each mouse and ascites formed in 7-14 days.Ascites fluid was harvested with a pasteur pipette through a hole in theperitoneum.

Reactivity of Monoclonal Antibodies

Monoclonals useful in this invention include those which are specificfor FIV and form a sufficiently strong interation with an FIV epitope,and an FIV antigen, to be useful in an immunoassay, for example, anELISA to detect FIV antigen. In order to determine which of the abovemonoclonal antibodies are useful in this invention two main tests wereused. The first was to determine whether the monoclonal antibody canbind FIV antigen and be detected with a conjugate of polyclonal antibodyto FIV (an ELISA test, described in detail below). The second test wasto form a conjugate of the monoclonal antibody with horseradishperoxidase and then determine whether the monoclonal antibody was ableto compete with itself for FIV antigen, and whether it would competewith other monoclonal antibodies for that antigen. The latter test isuseful to determine which monoclonals are useful in a mixture ofmonoclonal antibodies; generally antibodies with different reactivitieswill be combined together to allow detection of a larger range of FIVantigens in a sample. Such combination will greatly increasesensitivity.

Another test is to perform a Western blot to determine whether themonoclonal antibody has good reactivity with one or more FIV antigens.Generally, those monoclonals which show poor reactivity, that is,produce faint bands on the Western blot, are not suitable in thisinvention. Yet another test involves radioimmunoprecipitation assay(RIPA) where FIV virus labeled with ³⁵ S-methionine is reacted with amonoclonal antibody to form within immunoprecipitate, and theimmunoprecipitate run in a SDS-PAGE and autoradiographed to detect thelabelled proteins. This analysis determines which of the monoclonalantibodies is able to detect precursor FIV proteins and not just matureproteins.

Examples of such tests are presented below, along with a description ofthe identification of various FIV antigens (or proteins) by which themonoclonal antibodies can be distinguished. These examples are notlimiting to this invention.

Referring to FIG. 1, proteins associated with purified FIV were analyzedby SDS/PAGE and compared with proteins isolated in an identical mannerfrom the spent culture medium of uninfected cells. Analysis of theCoomassie Blue stained gels revealed three major proteins with molecularweights of about 10, 15, and 26 kD, named p10, p15 and p26,respectively.

When an ELISA test was performed using disrupted FIV to identify catspossessing polyclonal antibody to FIV proteins, and Western blotanalysis then performed on feline sera determined to be positive byELISA, each of the cats had antibodies which reacted with one or moreproteins or antigens of molecular weight p10, 15, 26, 40 and 65 kD underthe conditions used.

Referring to FIG. 2, a standard Western immuno blot was performed asdescribed by Towbin et al., 76 Proc. Natl. Acad. Sci USA 4350, 1979.Briefly, FIV was disrupted with SDS and proteins transferred to a sheetof nitrocellulose. The nitrocellulose sheet was blocked with 30% calfserum, 1% bovine serum albumin (BSA), and 0.05% Tween 20 in Dulbecco'sphosphate buffer saline. The sheets were cut into 0.5 cm strips andincubated with a 1:100 dilution of serum sample in blocking buffer for 2hrs. for 20°-22° C. Strips were repeatedly washed with washing buffer(0.05% Tween 20 in Dulbecco's phosphate buffer saline) and thenincubated with a second antibody (specific for feline heavy and lightchain Ig) horseradish peroxidase conjugate (obtained from Kirkguard andPerry Laboratories Inc. Gaithersburg, MD). After 1 hr. incubation, thestrips were repeatedly washed with washing buffer and incubated with theprecipitating substrate 4-chloronaphthol for 10 min. The strips werepartially dried and the results interpreted immediately. The serum ineach of the lanes A-G was obtained from various cats infected with FIV.Predominant reactivity is detected with p. 26 and p15 and to a lesserextent with p10. Other proteins of 32, 40, 47 and 65 kD molecular weightare also detected.

Antibodies to glycoproteins can also be isolated. In particular, twoglycoproteins of molecular weight 40 kD (gp40) and 130 kD (gp130) can bedetected using PAGE and RIPA respectively.

Certain viral proteins, such as the qaq (p26) antigens, are abundant inpurified viral preparations, others such as the viral envelope proteins(gp130) tend to be lost during viral purification, and electrotransferless efficiently for Western blot analysis than the qaq antigens.Therefore, in order to more readily detect the viral envelope and theqaq precurser proteins FIV cell extracts were labeled with ³⁵Smethionine and cysteine and examined by immunoprecipitation (RIPA).Confluent cultures of cells infected with FIV were incubated for 30 min.in methionine and cysteine free Dulbecco's modified Eagle's medium. Thecell cultures were then incubated for 4 hrs. in 8 ml of the same mediumcontaining 100 microCuries per ml of ³⁵ S-methionine and ³⁵ S cysteine(specific activity 1200 Curies per mM, New England Nuclear Corporation,Boston, MA). The radioactive tissue culture fluids were removed and thecells lysed with 5 ml of 10 mM sodium phosphate buffer pH 7.5 containing100 mM NaCl, 1% Triton×100, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mMphenylmethylsulfonylfluoride, and 100 Kallikren inactivator units ofaprotenin per ml. (Sigma Chemical Co., St. Louis, MO). Before use, thecell lysates were clarified by centrifugation 100,000× g for 30 min. andthe pellet discarded. Aliquots of the labelled cell lysates (0.1 ml) and5 μl of serum being tested were mixed in a microcentrifuge tube andincubated for 1 hr. at 37° C. and then overnight at 4° C. The next day,0.2 ml of a 5% suspension of protein A Sepharose CL 4B beads (Pharmacia,Piscataway, NJ) in 10 mM of phosphate buffer, pH 7.5 containing 100 mMNaCl, 1% Triton X-100 and 0.1% SDS was added to each tube and mixed for30 min. at 4° C. The antibody/antigen complexes bound to the protein ASepharose beads were collected by centrifugation (2 min. at 20,000× g)and washed 3 times in lysing buffer. The final pellet was resuspended in25 μl SDS/PAGE loading buffer and heated and 100° C. for 3 minutes. TheSepharose beads were removed by centrifugation and the supernatantapplied to a PAGE. Gels were processed for fluorography usingenlightening™ (New England Nuclear Corporation, Boston, MA) and exposedat -70° to Kodak XR-5 film. Sera from experimentally infected catsrecognize proteins of 15, 22, 36, 40, 47, 110 and 130 kD. Although therewere some quantitative and qualititative differences all cats appear tomount a response to p22, p40, gp47 and gp130.

In order to determine which of the proteins identified by RIPA-PAGEanalysis were related to the major internal structural protein, p26,RIPA-PAGE analysis was carried out using monoclonal antibodies whichreacted with p26as determined by Western blotting. This monoclonalimmunoprecipited proteins p47, p36, p22 and p15. The high molecularweight 130 KD protein of FIV was not detected by the p26 specificmonoclonal antibody. A protein of molecular weight 100 kD was alsodetectable utilizing serum antibodies obtained from some infected cats.

The monoclonals isolated as described above and tested by the abovetechniques were found to react specifically with p10, p15, p26, p47 andto belong to the subtypes IgM, IgG1, IgG2, IgG3, IgG2A, and IgG2B. Ofthese, some monoclonals reacted poorly with p26 and p47 and othersreacted strongly or at an intermediate level. We found that the mostsignificant test for usefulness of any specific antibody was the ELISAtest. In a specific test, microtiter plates were coated with theanti-FIV monoclonal antibodies at 1 microgram per well in coating buffer(0.1 M NaHPO₄ pH 7.2). The monoclonal antibodies were incubated at 3° C.overnight The plates were then washed two times with phosphate bufferedsaline and 0.05% Tween-20 and the remaining liquid aspirated. The wellswere then blocked with 1% bovine serum albumin in 0.1 M Tris pH 7 andthis wash solution aspirated. FIV sample or a known FIV-antigen was thenadded by standard procedure. Horseradish peroxidase conjugate wasprepared using either purified goat anti-FIV IgG or a second anti-FIVmonoclonal antibody. Conjugate was then provided to the test wells.Following washing, a substrate-chromogen mixture (TMB/H₂ O₂) was addedto test wells and incubated for 15 minutes. Optical densities of wellsat 650 nm was then determined. In this test a normal negative gave aresult of approximately 0.06 O.D. units. A poor monoclonal antibodyprovided with 1 μg/ml of FIV antigen gave an O.D. reading of less than0.5, cultures having a fairly good antibody yielded O.D. readings of 0.5to 1.0. A good monoclonal antibody typically yielded an O.D. of greaterthan 0.2 when 1 ng per ml FIV was provided.

In a second experiment, each of the monoclonal antibodies was conjugatedwith horseradish peroxidase and analyzed in a competition assay for FIVantigen. In this way it was determined which monoclonal antibodies couldbe used in mixtures for standard immunoassays for FIV. Particularly goodmixtures included the mixture of 2D4 or 3H8 (specific for p26), 4F2(specific for p15, p26 and p50) and 6E6 (specific for p26 and p-50).

Deposits

Cell lines producing monoclonal antibodies 3H8, 4F2 and 6E6 weredeposited with ATCC on Nov. 1, 1988 and given designation numbersHB9888, HB9889 and HB9890, respectively.

Applicants' assignee, Idexx Corp., acknowledges its responsibility toreplace these cultures should they die before the end of the term of apatent issued hereon, 5 years after the last request for a culture, or30 years, whichever is the longer, and its responsibility to notify thedepository of the issuance of such a patent, at which time the depositswill be irrevocably made available to the public. Until that time thedeposits will be made available to the Commissioner of Patents under theterms of 37 CFR §1-14 and 35 USC §112.

Other embodiments are within the following claims.

We claim:
 1. A monoclonal antibody specific for an epitope of the felineimmunodeficiency virus, FIV encoded antigen
 10. 2. A monoclonal antibodyspecific for an epitope of the FIV-encoded antigen p110.
 3. A monoclonalantibody specific for an epitope of the FIV-encoded antigen gp130.
 4. Acomposition comprising at least two monoclonal antibodies, each antibodybeing specific for a different epitope of an FIV-encoded antigenicprotein, said antigenic protein being chosen from the group consistingof p10, p15, p26, p47, p110, gp40, gp130.
 5. A method for detection ofan epitope of an FIV-encoded antigenic protein in a sample, saidantigenic protein being chosen from the group consisting of p10, p15,p26, p110, gp40, gp130, said method comprising the steps of:a) providinga monoclonal antibody specific for said epitope; b) contacting saidantibody with said sample under conditions in which said antibody formsa complex with said epitope; and c) detecting said complex, wherein thepresence of said complex indicates the presence of said epitope in saidsample.